By activity-guided fractionation, gliotoxin was isolated as an antibacterial metabolite of the fungus Penicillium decumbens which was derived from the jellyfish Nemopilema nomurai. Gliotoxin was further evaluated for antibacterial activity against several piscine and human MDR (multidrug resistance) pathogens. Gliotoxin showed significant antibacterial activity against Gram-positive piscine pathogens such as Streptococcus iniae FP5228, Streptococcus iniae FP3187, Streptococcus parauberis FP3287, Streptococcus parauberis SPOF3K, S. parauberis KSP28, and Lactococcus garvieae FP5245. Gliotoxin showed strong activity especially against S. parauberis SPOF3K and S. iniae FP5228, which are resistant to oxytetracycline. It is noteworthy that gliotoxin effectively suppressed streptococci which are the major pathogens for piscine infection and mortality in aquaculture industry. Gliotoxin also showed strong antibacterial activity against multidrug-resistant human pathogens (MDR) including Enterococcus faecium 5270 and MRSA (methicillin-resistant Staphylococcus aureus) 3089.
Aquaculture ; Enterococcus faecium ; Fungi ; Gliotoxin ; Humans ; Lactococcus ; Methicillin-Resistant Staphylococcus aureus ; Mortality ; Oxytetracycline ; Penicillium ; Staphylococcus ; Streptococcus

Aspergillus funigatus and other pathogenic fungi synthesize a toxic epidithi- odiopiperzine (ETP) metabolite called gliotoxin. Gliotoxin is an epidithiodiopiperzine compound which can both react with sulfhydryl groups and form hydrogen peroxide. The fungal toxin gliotoxin induces apoptotic cell death in a variety of cells. Apoptosis induced by gliotoxin need calcium but effect of calcium preconditioning is unknown by gliotoxin. We studied the effect of protein kinase C and calcium preconditioning on gliotoxin-induced apoptosis in H9c2 cell. PKC and calcium preconditiong inhibited DNA fragmentation by gliotoxin. From this above results suggest that gliotoxin induce apoptosis via caspase-3 activation, because caspase-3 inhibitor (DEVD-CHO) didn't induce apoptosis in gliotoxin treated H9c2 clls. Calcium and PKC preconditioning inhibit caspase-3 activation by gliotoxin. These data means that PKC preconditioning is related with caspase-3 regulate in gliotoxin-induced apoptosis.
Apoptosis* ; Aspergillus ; Calcium ; Caspase 3 ; Cell Death ; DNA Fragmentation ; Fungi ; Gliotoxin* ; Hydrogen Peroxide ; Protein Kinase C* ; Protein Kinases*

PURPOSE: Cell therapy for various diseases has gained wide acceptance. Because most patients with chronic liver failure have mild-to-severe liver cirrhosis, there are many limitations to clinical applications. We analyzed how to increase cell engraftment in rats with liver fibrosis. METHODS: We used analbuminemic SD rats (NARs) as recipients of syngeneic CAG-EGFP SD hepatocytes obtained by the 2 perfusion method. Hepatic fibrosis was induced with thioacetamide in drinking water for 6 weeks in the recipient NARs. NARs were pre-treated with gadolinium, doxorubicin, and gliotoxin before hepatocyte transplantation. We evaluated the degree of cell engraftment by RT-PCR and immunofluorescent staining for GFP and albumin. The transplanted cells were detected by immunostaining for albumin, and serum albumin was also measured. RESULTS: Although detection of GFP by RT-PCR was variable, albumin was detected in all groups 4 wks after hepatocyte transplantation. GFP and albumin were also detected by immunofluorescent staining 1 and 4 wks after cell transplantation. In control rats, albumin production was maximal at 3 wks, and after that it rapidly decreased. In the gadolinium and doxorubicin-treated group, albumin production was increased up to 4 wks. Albumin production in the gadolinium-treated group was superior to that of the doxorubicin-treated group. CONCLUSION: Kupffer cells play the most important role in cell engraftment in hepatic fibrosis. Therefore, perturbation of kupffer cells in hepatic fibrosis is needed to increase cell engraftment.
Animals ; Cell Transplantation ; Doxorubicin ; Drinking Water ; End Stage Liver Disease ; Fibrosis ; Gadolinium ; Gliotoxin ; Hepatocytes ; Humans ; Kupffer Cells ; Liver ; Liver Cirrhosis ; Perfusion ; Rats ; Serum Albumin ; Thioacetamide ; Tissue Therapy ; Transplants

PURPOSE: Cell therapy for various diseases has gained wide acceptance. Because most patients with chronic liver failure have mild-to-severe liver cirrhosis, there are many limitations to clinical applications. We analyzed how to increase cell engraftment in rats with liver fibrosis. METHODS: We used analbuminemic SD rats (NARs) as recipients of syngeneic CAG-EGFP SD hepatocytes obtained by the 2 perfusion method. Hepatic fibrosis was induced with thioacetamide in drinking water for 6 weeks in the recipient NARs. NARs were pre-treated with gadolinium, doxorubicin, and gliotoxin before hepatocyte transplantation. We evaluated the degree of cell engraftment by RT-PCR and immunofluorescent staining for GFP and albumin. The transplanted cells were detected by immunostaining for albumin, and serum albumin was also measured. RESULTS: Although detection of GFP by RT-PCR was variable, albumin was detected in all groups 4 wks after hepatocyte transplantation. GFP and albumin were also detected by immunofluorescent staining 1 and 4 wks after cell transplantation. In control rats, albumin production was maximal at 3 wks, and after that it rapidly decreased. In the gadolinium and doxorubicin-treated group, albumin production was increased up to 4 wks. Albumin production in the gadolinium-treated group was superior to that of the doxorubicin-treated group. CONCLUSION: Kupffer cells play the most important role in cell engraftment in hepatic fibrosis. Therefore, perturbation of kupffer cells in hepatic fibrosis is needed to increase cell engraftment.
Animals ; Cell Transplantation ; Doxorubicin ; Drinking Water ; End Stage Liver Disease ; Fibrosis ; Gadolinium ; Gliotoxin ; Hepatocytes ; Humans ; Kupffer Cells ; Liver ; Liver Cirrhosis ; Perfusion ; Rats ; Serum Albumin ; Thioacetamide ; Tissue Therapy ; Transplants

Gliotoxin, a fungal metabolite, is one of the epipolythiodioxopiperazine classes and has a variety of effects including imrnunomodulatory and apoptotic agents. This study is designed to evaluate the effect of zinc on gliotoxin-induced death of HL-60 cells. Here, we demonstrated that treatment of gliotoxin decreased cell viability in a dose and time-dependent manner. Gliotoxin-induced cell death was confirtned as apoptosis characterized by chromatin marginafion, fragmentation and ladder-pattern digestion of genomic DNA. Gliotoxin increased the proteolytic activities of caspase 3, 6, 8, and 9. Caspase-3 activation was further confirmed by the degradation of procaspase-3 and PARP in gliotoxin-treated HL-60 cells. Zinc compounds including ZnC12 and ZnSO4 markedly inhibited gliotoxin-induced apoptosis in HL-60 cells (from 30% to 90%). Consistent with anti- apoptotic effects, zinc also suppressed the enzymatic activities of caspase-3 and -9 proteases. In addition, cleavage of both PARP and procaspase 3 in gliotoxin-treated HL-60 cells was inhibited by the addition of zinc compounds. We further demonstrated that expression of Fas ligand by gliotoxin was suppressed by zinc compounds. These data suggest that zinc may prevent gliotoxin- induced apoptosis via inhibition of Fas ligand expression as well as suppression of caspase family cysteine proteases-3 and -9 in HL-60 cells.
Apoptosis* ; Caspase 3 ; Cell Death ; Cell Survival ; Chromatin ; Cysteine ; Digestion ; DNA ; Fas Ligand Protein ; Gliotoxin* ; HL-60 Cells ; Humans ; Peptide Hydrolases ; Zinc Compounds ; Zinc*

Aspergillus funigatus and other pathogenic fungi synthesize a toxic epidithi-odiopiperzine (ETP) metabolite, namely gliotoxin. Gliotoxin commonly react with sulfhydryl groups, and then, forms hydrogen peroxide. These fungal toxins induce apoptotic cell death in various cells. Apoptosis induced by gliotoxin need calcium. Effect of calcium preconditioning was not reported in gliotoxin-induced apoptosis. To examine the effect of protein kinase C (PKC) and calcium which was regulate caspase-3, PKC and calcium preconditioning before gliotoxin treatment, apoptotic agents such as bcl-2 family, caspase-3 and DNA fragmentation in A7r5 cell line from rat smooth muscle cell were studied. These results showed that gliotoxin induces the expression of bad of bcl-2 family, caspase-3 activation and DNA fragmentation in A7r5 cells. Gliotoxin treatment followed by calcium and PKC preconditioning suppress the Bad of bcl-2 family, and inhibited caspase-3 activation, respectively. These results suggest that PKC and calcium preconditioning protect the gliotoxin-induced apoptosis, through the protection of pro-apoptotic bcl-2 family in A7r5 cells.
Animals ; Apoptosis ; Aspergillus ; Calcium ; Caspase 3 ; Cell Death ; Cell Line ; DNA Fragmentation ; Fungi ; Gliotoxin ; Humans ; Hydrogen Peroxide ; Muscle, Smooth* ; Mycotoxins ; Myocytes, Smooth Muscle* ; Protein Kinase C ; Rats*

Members of the genus Aspergillus are the most common fungi and all reproduce asexually by forming long chains of conidiospores (or conidia). The impact of various Aspergillus species on humans ranges from beneficial to harmful. For example, several species including Aspergillus oryzae and Aspergillus niger are used in industry for enzyme production and food processing. In contrast, Aspergillus flavus produce the most potent naturally present carcinogen aflatoxins, which contaminate various plant- and animal-based foods. Importantly, the opportunistic human pathogen Aspergillus fumigatus has become the most prevalent airborne fungal pathogen in developed countries, causing invasive aspergillosis in immunocompromised patients with a high mortality rate. A. fumigatus produces a massive number of small hydrophobic conidia as the primary means of dispersal, survival, genome-protection, and infecting hosts. Large-scale genome-wide expression studies can now be conducted due to completion of A. fumigatus genome sequencing. However, genomics becomes more powerful and informative when combined with genetics. We have been investigating the mechanisms underlying the regulation of asexual development (conidiation) and gliotoxin biosynthesis in A. fumigatus, primarily focusing on a characterization of key developmental regulators identified in the model fungus Aspergillus nidulans. In this review, I will summarize our current understanding of how conidiation in two aspergilli is regulated.
Aflatoxins ; Aspergillosis ; Aspergillus ; Aspergillus flavus ; Aspergillus fumigatus ; Aspergillus nidulans ; Aspergillus niger ; Aspergillus oryzae ; Developed Countries ; Food Handling ; Fungi ; Genome ; Genomics ; Gliotoxin ; Humans ; Immunocompromised Host ; Spores, Fungal ; Transcription Factors

Many fungi including Penicillium, Aspergillus, Gliocladium, and Thermoascus produce an epipolythiodioxopiperazine class of fungal metabolite, gliotoxin, which contirbutes the pathogenesis of fungal infection as an immunomodulator and cytotoxic agent. This study is designed to define the mechanism by which gliotoxin exerts the cytotoxic effect of gliotoxin on human promyelocytic leukemic cells, HL-60. Gliotoxin induces the apoptosis of HL-60 cells which is characterized by the ladder pattern fragmentation of DNA. Gliotoxin induces the activation of DEVD-specific cysteine protease in a time- and dose-dependent rnanner. It also increases the phosphotransferase activities of c-Jun N-terminal kinase1 (JNK1) and p38 in gliotoxin-treated HL-60 cells. Furthermore, gliotoxin decreases the activation of transcriptional activator, actiating protein (AP-1) and NF-kB. These results suggest that gliotoxin induces the apoptotic death of HL-60 cells via activation of DEVD- specific caspase as well as mitogen activated protein kinases (MAP kinases) including JNK1 and p38, and inhibition of transcriptional activators, AP-1 and NF-kB.
Apoptosis* ; Aspergillus ; Caspase 3 ; Cysteine Proteases ; DNA ; Fungi ; Gliocladium ; Gliotoxin* ; HL-60 Cells* ; Humans ; Mitogen-Activated Protein Kinases ; NF-kappa B ; Penicillium ; Thermoascus ; Transcription Factor AP-1 ; Transcription Factors

During inflammation of the colon, cells of the gut mucosa express numerous inflammatory mediators including interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta(IL-1beta). These cytokines have been implicated as contributing factors in the inflammatory process, which may result in colitis during inflammatory bowel disease (IBD). Gliotoxin is a fungal metabolite of an epipolythiodioxopiperazine analogue with immunosup-pressive properties in vivo and in vitro, but the effects of gliotoxin on IBD have not been largely evaluated. Therefore, this study evaluated the potential of gliotoxin to protect against TNBS-induced colitis. One microgram of gliotoxin in 100microliter of vehicle was intra-rectally administered into mice exhibiting trinitrobenzene sulfonic acid (TNBS)-induced colitis. IL-8 secretion was measured using an enzyme-linked immu-nosorbent assay (ELISA), myeloperoxidase (MPO) activity was evaluated spectrophotometically, and IkappaB degradation was analyzed on Western blots. Gliotoxin treatment of mice bearing TNBS-induced colitis improved macro-and micro-pathological findings and dramatically decreased MPO activity, a marker of leukocyte infiltration. Furthermore, gliotoxin decreased IkappaB degradation and IL-8 induction caused by TNF-alpha or IL-1beta in HT-29 cells. These findings suggest that gliotoxin partially protects against TNBS-induced colitis through the sup-pression of IL-8 induction and IkappaB degradation by inflammatory mediators such as TNF-alpha or IL-1beta.
Animals ; Blotting, Western ; Colitis* ; Colon ; Crohn Disease ; Cytokines ; Down-Regulation* ; Gliotoxin* ; HT29 Cells ; Humans ; Inflammation ; Inflammatory Bowel Diseases ; Interleukin-8 ; Leukocytes ; Mice ; Mucous Membrane ; Peroxidase ; Tumor Necrosis Factor-alpha